Bison Bonasus

Annals of Agricultural and Environmental Medicine ORIGINAL ARTICLE www.aaem.pl

Microbiological and molecular monitoring for ONLINE FIRST bovine tuberculosis inONLINE the Polish population FIRST of European bison (Bison bonasus) Anna Didkowska1,A-F , Blanka Orłowska1,A-C,F , Monika Krajewska-Wędzina2,A,C,F , Ewa Augustynowicz-Kopeć3,A-C,F , Sylwia Brzezińska3,B-C,E-F , Marta Żygowska1,A-B,D,F , Jan Wiśniewski1,B-C,F , Stanisław Kaczor4,B,F , Mirosław Welz5,A,C,F , Wanda Olech6,A,E-F , Krzysztof Anusz1,A-F 1 Department of Food Hygiene and Public Health Protection, Institute of Veterinary Medicine, University of Life Sciences (SGGW), Warsaw, Poland 2 Department of Microbiology, National Veterinary Research Institute, Puławy, Poland ONLINE FIRST ONLINE3 FIRST Department of Microbiology, National Tuberculosis Reference Laboratory, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland 4 County Veterinary Inspectorate, Sanok, Poland 5 General Veterinary Inspectorate, Warsaw, Poland 6 Department of Animal Genetics and Conservation, Institute of Animal Sciences, University of Life Sciences (SGGW), Warsaw, Poland A – Research concept and design, B – Collection and/or assembly of data, C – Data analysis and interpretation, D – Writing the article, E – Critical revision of the article, F – Final approval of article Didkowska A, Orłowska B, Krajewska-Wędzina M, Augustynowicz-Kopeć E, Brzezińska S, Żygowska M, Wiśniewski J, Kaczor S, Welz M, Olech W, Anusz K. Microbiological and molecular monitoring for bovine tuberculosis in the Polish population of European bison (Bison bonasus). Ann Agric Environ Med. doi: 10.26444/aaem/130822 Abstract Introduction and objective. In recent years, bovine tuberculosis (BTB) has become one of the major health hazards facing the European bison (EB, Bison bonasus), a vulnerable species that requires active protection, including regular and effective health monitoring. Monitoring of zoonotic disease in wildlife is also an important part of public health protection. The aim of the study was to determine whether BTB still influences the EB population in Poland. Materials and method. During 2017–2019, mandibular, retropharyngeal and mediastinal lymph nodes were collected from 90 EB during post-mortem examination, and then cultivated on Lowenstein-Jensen and Stonebrink media. Isolated strains were subjected to molecular analysis to determine the species, spoligotype and MIRU-VNTR pattern. Results. Lesions were found in lymph nodes originating from eight EB (8.89%). Positive microbiological cultures for mycobacteria were obtained in samples from six (6.67%) EB. The isolated strains were identified as Mycobacterium caprae (material from four EB) and atypical mycobacteria (material from two EB). For M. caprae strains spoligotype M. bovis 4_CA 1600 was identified and the MIRU-VNTR pattern was identified as 345751355413232. Conclusions. It is recommended that this potentially dangerous disease should be monitored in EB via a comprehensive strategy based on a combination of microbiological and molecular methods. Such monitoring will protect the health of both animals and humans. Key words monitoring, bovine tuberculosis, Mycobacterium caprae, European bison, Mycobacterium avium complex

INTRODUCTION category in the International Union for Conservation (IUCN) Red Book of Endangered Species, its population is steadily Almost a quarter of the global European bison (EB, Bison growing (http://www.iucnredlist.org) [1]. The main goal of bonasus) population lives in Poland. The European bison EB restitution now is to increase the number of free-ranging Pedigree Book indicates that in 2018, the EB population in and enclosed-breeding herds [2, 3]. In Poland, the national Poland comprised 1,820 individuals: 207 – closed farms, EB protection project places a high priority on monitoring 1,613 – free-ranging herds (https://bpn.com.pl/index. the health of the population [4]. Such monitoring is primarily php?option=com_content&task=view&id=1132&Itemid=3 based on post-mortem examination, during which clinical 06]). EB is a priority species for the European Union in terms material is collected for further diagnosis [5]. of conservation measures (species code: 2674), and despite Suspsceptibilty to infectious diseases seems to be one EB being included in the VU (vulnerable to extinction) of the most crucial problems but there are also existing environmental threats [6]. In the 20th Century, a number of Address for correspondence: Anna Didkowska, Department of Food Hygiene and diseases affected the EB population in Poland, with the most Public Health Protection, Institute of Veterinary Medicine, University of Life Sciences (SGGW), Nowoursynowska 166, 02-787 Warsaw, Poland significant ones being foot-and-mouth disease, posthitis and Email: [email protected] bovine tuberculosis (BTB) [7]. BTB, caused by Mycobacterium Received: 15.19.2020; accepted: 24.11.2020; first published: 10.12.2020 bovis and Mycobacterium caprae, is a chronic disease that has AAEM Annals of Agricultural and Environmental Medicine Anna Didkowska, Blanka Orłowska, Monika Krajewska-Wędzina, Ewa Augustynowicz-Kopeć, Sylwia Brzezińska, Marta Żygowska, et al. Microbiological and molecular…

been found in many species of both livestock and wildlife [8, (Non-tuberculosis mycobacteria) species using the 9], including the genus Bison [10]. Not only is it important GenoType®Mycobacterium CM test (Hain Lifescience). Within that endangered animals be monitored for BTB, but as the the MTBC group, the species were further differentiated using ONLINE ®FIRST ONLINE FIRST disease is known to have zoonotic potential, its presence in GenoType MTBC assay (Hain Lifescience) which is based on other species also needs to be tracked [11, 12]. Even though, the polymorphism of the gyrB gene. GenoType tests are based currently, Poland has the officially tuberculosis free (OTF1) on DNA-STRIP method which can identify PCR products status, in recent years cases of M. bovis and M. caprae by allowing them to hybridize with specific oligonucleotide infection has been confirmed in wildlife [13, 14]. Sharing probes on a nitrocellulose strip. Both tests were carried out pastures by free-living animals with livestock can lead to in accordance with the manufacturer’s instructions. The the pathogens transmission and pose a real threat to public isolated DNA was also subjected to spoligotyping using a health. Wherefore, the monitoring of BTB in EB is important commercial genotyping kit (Genutaur molecular products, for two reasons – species protection and its zoonotic nature. Kampenhout, Belgium) (https://gentaur-spain.com/wp- Currently, despite the appearance of many new methods, content/uploads/2015/03/Spoligotyping-Manual.pdf). This the current gold standard in tuberculosis diagnosis is based method allows the polymorphism of the chromosomal DR on a combination of culture and molecular methods. These (direct repeat) region occurring only in the genome of MTBC ONLINEmethods areFIRST also the most appropriate for use in post- to be detected. The obtained patterns were compared to those ONLINE FIRST mortem monitoring. In such cases, the predilection tissue of strains registered in the SpolDB4 database. for mycobacteria isolation is that of the lymph nodes [15, 16]. In addition, the DNA obtained for each strain was subjected to 15 amplification reactions using 15 pairs of primer sequences based on the MIRU-VNTR method. This OBJECTIVES method is based on the analysis of selected polymorphic microsatellite sequences within the mycobacterial genome. As the severity of the threat presented to European bison in The PCR products were visualized by gel electrophoresis Poland by BTB remains unclear, the aim of the present study (2.5% agarose gel, large gel-65V, 5 hours). The following was to determine the occurrence of mycobacteria within loci were analyzed: MIRU-4, MIRU-10, MIRU-16, MIRU- the population by examination of lymph nodes collected 26, MIRU-31, MIRU-40, VNTR 424, VNTR 577, VNTR post-mortem from EB for BTB testing. The study also uses a 2165, VNTR 2401, VNTR 3690, VNTR 4165, VNTR 2163b, combination of microbiological and molecular methods to VNTR 1955, VNTR 4052. The results are presented as a classify any identified mycobacteria. 15-digit code. The digits indicate the number of repetitions of subsequent MIRU-VNTR repeat sequences.

MATERIALS AND METHOD RESULTS Material collection. During 2017–2019, lymph nodes were collected from 90 European bison (EB) from various regions Assessment of lesions in lymph nodes. Lesions were of Poland. The age of the EB ranged from three months to observed in lymph nodes originating from eight EB (8.89%), 25 years (mean age: 7.5 years). The ages of nine EB were among which four EB demonstrated caseous lesions and four unknown. Of the studied EB, 44.44% were from free-ranging presented purulent lesions. herds (Knyszyńska Forest, Bieszczady Mountains, Borecka Forest, Białowieska Forest) and 55.56% from breeding Mycobacteria isolation. Material obtained from five EBs centres. Post-mortem examination was performed on all demonstrated growth on both types of media, manifested as EB, during which lymph nodes (mandibular, retropharyngeal rough, yellow colonies on the surface of the medium. Material and mediastinal) were collected for further examination. The from one EB was found to give rise to two types of colony. nodes were delivered to the laboratory under refrigerated conditions and kept at -20 °C until cultures were performed. Genetic analysis. Of the seven tested strains, four were classified as M. caprae, two as Mycobacterium xenopii and one Mycobacteria isolation. The collected lymph nodes were as Mycobacterium avium. Further analysis of M. caprae found examined for the presence of anatomopathological lesions, the spoligotype for these strains to be identifiedM. caprae with particular emphasis on tuberculous lesions. Following – spoligotype M. bovis _4_ CA 1600 (octagonal pattern: this, the material was subjected to standard methods for 200003770003600) (SpolDB4 database). For the studied mycobacterial isolation according to Orlowska et al. (2017) M. caprae strains, the MIRU-VNTR pattern was identified [13]. Briefly, the material was homogenized in 5% oxalic acid as 345751355413232. and flushed twice in saline solution. The supernatant was then plated on Lowenstein and Stonebrink media, and incubated at 37oC for 12 weeks, with the growth on the media being DISCUSSION checked every seven days. BTB cases in EB should be should be carefully analyzed and Genetic analysis. DNA was isolated using the Genolyse monitored. In this study, 4.44% of tested samples were found isolation kit (Hain Lifescience, Germany). Based on to be BTB positive. These samples were obtained from EB the obtained DNA, the strains were classified as MTBC kept in an enclosed breeding centre where BTB had been (Mycobacterium tuberculosis complex) or as 27 NTM diagnosed previously. Although cases of BTB have been noted almost every year in this herd since 2013 [17], no new 1. OTF – officialy tuberculosis free. BTB outbreaks occurred in EB elsewhere in Poland in the Annals of Agricultural and Environmental Medicine AAEM Anna Didkowska, Blanka Orłowska, Monika Krajewska-Wędzina, Ewa Augustynowicz-Kopeć, Sylwia Brzezińska, Marta Żygowska, et al. Microbiological and molecular…

period 2017–2019. However, as BTB cases have been recorded humans by coming into contact with livestock. For Poland in EB in Poland in previous years, it is recommended that to maintain the OTF status, the prevalence of BTB in free- ONLINE FIRST lymph nodes from each dead EB be collected post mortemONLINE ranging animals, especially the EB and wild boarsFIRST of the and subjected to mycobacterial examination. Bieszczady Mountains, needs to be effectively monitored. European bison appear to be particularly sensitive to BTB The possible loss of the OTF status would be associated infection, as indicated by the large number of cases observed with economic losses and trade restrictions, therefore it is in the past [18]. In the period 1996–2013, 45 cases of BTB were of a great importance to monitor BTB, especially in highly confirmed in EB Bison( bonasus caucasicus) in the Bieszczady sensitive species. Mountains in south-east Poland [18]. The first case concerned M. caprae have been isolated previously in European a three-year-old cow in which generalized tuberculosis was wildlife such as red deer (Cervus elaphus) [41, 42, 43, 44], found during post-mortem examination [19]. Following this, red fox (Vulpes vulpes) [45] and wild boar [46]. It has also in the period 2005–2008, BTB was confirmed in two EB from been described in wild boar (Sus scrofa), roe deer (Capreolus the Bieszczady area [20]. In 2009, an entire free-ranging capreolus) and wolves (Canis lupus) in Poland [47]. The MIRU- herd in the Bieszczady Mountains was culled following the VNTR pattern obtained in the present study has not been discovery of BTB: tuberculosis lesions were found during described previously. It may be of great importance in the case ONLINE FIRST ONLINEpost-mortem examination in all herd members, andFIRST BTB was of outbreaks of tuberculosis in European bison, or in other confirmed microbiologically in 23 out of 24 individuals [21]. wildlife or livestock animals, especially those from the same It should be noted that since 2012, BTB cases have also region. Such information can help establish transmission been reported in wild boars in the Bieszczady Mountains. routes and allow epidemiological investigations [48]. Interestingly, the strain identified in the boars demonstrated It is notable that atypical mycobacteria were isolated from an identical spoligotype to a herd in which BTB had previously the EB in the present study. Even though M. avium infection been diagnosed in 13 out of 18 individuals [14]; although in animals can be asymptomatic, their occurrence should be the decision was made to cull the entire herd, several EB monitored in protected species as infection is often associated separated from the herd and could not be found [22]. The with loss of condition, and the occurrence of diarrhea and long history of BTB in the Bieszczady Mountains justifies weight loss [49]. In addition, the isolated mycobacteria greater monitoring for BTB, particularly in this region. In (M. avium and M. xenopi) tend to be etiological factors of addition, in 2016, two cases of BTB were also confirmed in serious diseases in humans, particularly those with immune free-ranging EB in a herd in the Borecka Forest [23] in the deficiency [50, 51, 52]. Warmia-Mazuria region of north-east Poland, but no cases where confirmed in this region later [24]. Attempts have been made to isolate mycobacteria ante- CONCLUSIONS mortem from animals in captivity, such as in zoos and breeding centres, using materials such as bronchopulmonary Microbiological monitoring in 2017–2019 indicated that lavage from lions [25] or EB [26]. However, due to the BTB does not currently pose a significant threat to the EB difficulties associated with collecting material, indirect population. However, among the samples taken from bison in tests are typically used for ante-mortem diagnostics in a breeding centrer where BTB had previously been detected, free-ranging animals, such as the tuberculin test [27, 28], 4.4% were found to be positive for bovine mycobacteria, and gamma-interferon test [29, 30, 31, 32] or serological tests 2.2% for atypical mycobacteria. No new BTB outbreaks were [33, 34, 35]. These tests, however, are less sensitive thanpost- detected in the period 2017–2018. Despite these negative mortem microbiological monitoring, and they often lack results, it is recommended that further microbiological standardization for different species, including EB. For this monitoring for BTB be performed in this species. reason, ante-mortem monitoring for BTB is not sufficient for monitoring the epizootic situation in the EB population, and Acknowledgments material obtained post-mortem remains extremely valuable. The work was supported by the project “Complex project One of the goals of sustainable development set by of European bison conservation by State Forests”, which the World Health Organization is to combat the global is financed by the Forest Found (Poland), contract no tuberculosis epidemic. It should be emphasized that data on OR.271.3.10.2017. the number of tuberculosis cases in humans are considered to be underestimated and the real scale of the problem remains unknown [36]. 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